CN109680026B - Purification of recombinant CA16 virus-like particles, application of recombinant CA16 virus-like particles in vaccine and vaccine - Google Patents
Purification of recombinant CA16 virus-like particles, application of recombinant CA16 virus-like particles in vaccine and vaccine Download PDFInfo
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Abstract
The invention provides a purification method of a recombinant CA16 virus-like particle, application in a vaccine and the vaccine. The recombinant expression engineering bacteria are subjected to high-density fermentation culture and methanol induced expression of CA16 virus particle protein, the bacteria are collected centrifugally and subjected to high-pressure homogenate crushing, and the supernatant is purified by ultrafiltration, ion exchange chromatography, hydroxyapatite chromatography, molecular sieve chromatography and the like to obtain the recombinant expression CA16 virus particle protein. The CA16 virosome vaccine provided by the invention has good immunogenicity, safety, immunological characteristics and biological activity, the process is simple, the purification adopts a chromatography method, the linear amplification is more facilitated compared with density gradient centrifugation, the large-scale preparation and purification can be realized, VLP stock solution with high purity (more than 99%) can be obtained, the VLP stock solution can be used for preparing the vaccine for preventing CA16 infection, and the economic value and the application prospect are good.
Description
Technical Field
The invention relates to the field of biological products, in particular to a purification method of recombinant CA16 virus-like particles and a vaccine prepared by using the purified CA16 virus-like particles.
Background
The hand-foot-and-mouth disease (HFMD) is a common infectious disease of children, takes skin herpes of hands and feet and oral mucosa rash as main clinical manifestations, most cases are self-limiting diseases, the herpes and the oral mucosa rash are relieved automatically within 5-7 days, few cases rapidly develop into severe cases, and a few cases can generate aseptic meningoencephalitis, brainstem encephalitis, neurogenic pulmonary edema, heart damage and the like.
The hand-foot-and-mouth disease is a global infectious disease, and the epidemic of the disease is reported in most areas of the world. In 1957, the first outbreak occurred in new zealand. In 1958 Robinson, canada isolated Coxsackie virus group A16 (CA 16) from the patient's faeces and from the pharyngeal swab. In the middle of the 70's of the 20 th century, successive outbreaks of bulgaria and hungary had a CA16 epidemic with the central nervous system as the main clinical feature, and only bulgaria reported 750 cases of morbidity, 149 paralytic and 44 deaths. The u.k.1994 developed an epidemic of hand-foot-and-mouth disease caused by CAl6 that spread all over the welsh of england in 4 quarters, and 952 cases were observed in the monitoring sentinel, the largest of the records from this country. Patients are mostly 1-4 years old. In 2000, HFMD outbreak occurred in Singapore, and 3790 cases of disease attack and 5 cases of death occur. In 2000, months 9 to 10, singapore reported a hand-foot-and-mouth disease of 3790, 5 deaths, with CA16 being the major pathogen.
Chinese posters in 1981 have reported hand-foot-and-mouth disease, and the pathogen is enterovirus 71 (EV 71). Thereafter, it was reported in Beijing, hebei, tianjin, fujian, shandong, etc. In 1983, xiamen developed an HFMD epidemic, and CA16 type virus was isolated from infant specimens. In the same year, the body fluid has the hand-foot-and-mouth disease caused by CA 16. In 1998, the foot-and-mouth disease epidemic caused by EV71 infection in Taiwan area of China has 13 ten thousand cases. In 2000, nearly 1700 cases of disease are caused by Shandong in 5-8 months. After 2008, the hand-foot-and-mouth disease has a tendency to spread. 3736 cumulative reported cases of Anhui Fuyang from 3 to 5 months 2008, 22 of which died. The disease is reported in most provinces of China.
In fact, various enteroviruses can cause hand-foot-and-mouth diseases, but the enterovirus 71 (EV 71) and the Coxsackie virus A group 16 (CA 16) are main pathogens of hand-foot-and-mouth diseases in recent years in China. CA16 is the main pathogen in the early stage of HFDM epidemic, but because its onset symptoms are relatively mild, it has not attracted much attention, and there is no vaccine for preventing CA 16. The coxsackie virus can cause various diseases after infecting people, including respiratory diseases, myocarditis, pericarditis, nervous system diseases and the like. And the main spread population of the hand-foot-and-mouth disease is invisible infectors, and most people are shown as invisible infection after enterovirus infection and are difficult to find clinically, so that effective isolation measures are difficult to develop and spread easily, and the development of vaccines has important significance for preventing the hand-foot-and-mouth disease.
Currently, the vaccines for podopathies are mainly inactivated vaccines, virus Like Particle (VLP) vaccines, subunit vaccines, DNA vaccines, live attenuated vaccines, and the Like. At present, vaccines for preventing hand-foot-and-mouth diseases are only sold on the market at home, belong to inactivated vaccines, and prevent the hand-foot-and-mouth diseases caused by EV 71; for the CA16 vaccine, studies are now in preclinical stage, mainly inactivated vaccines, subunit vaccines, DNA vaccines and live attenuated vaccines. The research is carried out more rapidly by inactivated vaccines, but the inactivated vaccines have toxic adverse effects, virus particles are destroyed during inactivation, and the difference of hollow solid particles causes batch-to-batch instability. Therefore, the development of a safer and more effective preventive vaccine aiming at CA16 has great significance for controlling the epidemic of the infant hand-foot-and-mouth disease.
The emergence of virus-like particle vaccines provides a new opportunity for the development of novel, safe and effective vaccines. Virus-like particles are hollow particles containing one or more structural proteins of a virus, are free of viral nucleic acids (DNA/RNA), cannot replicate autonomously, are identical or similar in morphology to authentic virus particles, and can be presented to immune cells in the same way as viral infection, effectively inducing an immunoprotective response in the body's immune system. The capsid proteins of the virus generally have a natural self-assembly capacity. The VLPs vaccine has no infectivity, good stability, difficult inactivation and wide development prospect.
The CA16 virus particle has an icosahedral spherical structure, is three-dimensionally symmetrical, has the diameter of about 23-30 nm, has no envelope and no protrusion, and consists of single-stranded positive-strand RNA and protein. The total length of the RNA chain is about 7400 nucleotides; the protein coat consists of 4 polypeptide chains, namely VP1, VP2, VP3 and VP4. These 4 proteins constitute a subunit, and 60 subunits constitute the capsid proteins of the virus. The capsid proteins of the virus generally have a natural self-assembly capacity. VLPs vaccine has no infectivity, good stability, difficult inactivation and wide development prospect.
The expression systems commonly used in virus-like particles at present are mainly prokaryotic expression systems and eukaryotic expression systems. Most of the proteins expressed by prokaryotic expression systems lose their native conformation and do not produce protective antibodies. Or the expression product is mostly inclusion body, the denaturation and renaturation steps of the inclusion body are complex, especially the expression of VLP, and the purification process is more complex. Examples of eukaryotic expression systems include mammalian cell expression systems, insect baculovirus expression systems, and yeast expression systems. The protein can spontaneously form VLP in a eukaryotic expression system, and great convenience is provided for a purification process. However, the VLP prepared by adopting insect cells has higher requirement on culture conditions and complex purification process, and the requirement of large-scale production is limited; in addition, the baculovirus-insect cell expression system produces baculovirus particles and other contaminants affecting the effect of the vaccine, and the baculovirus particles are difficult to separate from the VLPs prepared and require measures such as inactivation treatment, thus having a great influence on the quality of the vaccine.
The hansenula polymorpha expression system has the characteristics of stable genetic property, simple operation, high-density fermentation culture, high yield of target products, low production cost, suitability for industrial large-scale production and the like, also has the advantages of foreign protein post-translational processing and the like which cannot be possessed by a prokaryotic expression system, and is an advanced VLP vaccine expression system superior to escherichia coli and other eukaryotic expression systems.
Disclosure of Invention
The invention aims to provide a purification method of CA16 virus particles expressed in a hansenula polymorpha expression system, which is more favorable for linear amplification, can be prepared and purified on a large scale, can obtain VLP stock solution with high purity (more than 99 percent), can be used for preparing vaccines for preventing CA16 infection, and has better economic value and application prospect.
The above object of the present invention is achieved by the following technical solutions: a method for purifying recombinant CA16 virus-like particles, comprising the following steps:
(1) Fermenting the recombinant Hansenula polymorpha engineering bacteria containing CA16 coat protein P1 genes and 3CD protease genes;
(2) Crushing engineering bacteria, clarifying and ultrafiltering a target product;
(3) Ion exchange chromatography;
(4) Chromatography on hydroxyapatite;
(5) And (4) performing molecular sieve chromatography.
By adopting the technical scheme, experiments show that the VLP stock solution with high purity (more than 99%) can be obtained by adopting the method and is used for preparing human vaccines. The invention adopts three-step chromatography, has simple process, high antigen recovery rate of more than 40 percent, strong controllability of production process, large-scale production and high social value and economic value.
Preferably: the engineering bacteria are re-suspended with cell lysis buffer solution and crushed under 1100-1400 bar pressure for 2-4 times; the cell lysis buffer is 20mM NaH 2 PO 4 ，2mM EDTA-Na 2 0.2-1.0M NaCl,2mM PMSF, 0.01-1.0% Tween-80, and pH6.8-7.4.
Preferably, the engineering bacteria are used for breaking cells for 2 times under the pressure of 1250 bar.
By adopting the technical scheme, a large number of experiments show that the breakage rate of the cells can reach 50-70% after the cells are broken once by 1250 Bar; the crushing rate can reach 85-95 percent after two times of crushing, the crushing rate can reach more than 90-95 percent after three times of crushing, and the crushing rate of the two times of crushing is preferably more than 85 percent in consideration of the crushing time cost and the stability effect of the protein after the crushing.
Preferably: the method for harvesting the clarified liquid of the target product in the step (2) comprises the following steps:
pouring the crushed cell sap into a centrifugal cylinder, centrifuging at 6000-8000 rpm for 40-60 min, and collecting clear liquid;
or filtering the crushed cell sap through a depth filter at the filtering flow rate of 925-1850 mL/min/m 2 Collecting the clear liquid;
preferably, the following components: pouring the crushed cell sap into a centrifuge bowl, centrifuging at 7000rpm for 40min, and collecting clear liquid.
Through adopting the technical scheme, a large number of experiments show that the cell crushing liquid in the application adopts a centrifugal rotating speed lower than 6000rpm, the crushed thallus fragments cannot be removed, and the processing capacity is reduced by replacing 500mL centrifugal cups with centrifuge rotors higher than 8000rpm, so that the fragment removing effect can be achieved by adopting the optimal centrifugal rotating speed of 7000rpm and 40min of 1L centrifugal cups.
Preferably, the following components: the ultrafiltration in the step (2) comprises the following steps: and (3) carrying out ultrafiltration on the collected supernatant by a membrane package with 100-500KD and adopting a buffer solution with pH of 7.5-8.5 to remove small molecular substances, and collecting an ultrafiltrate to obtain a crude pure protein solution, wherein the components and the molar ratio of the buffer solution are as follows:
2-5 parts of trihydroxymethyl aminomethane
15 to 30 portions of NaCl
The buffer solution also comprises a glycerol aqueous solution with the mass fraction of 0-10%.
Preferably, the following components: the buffer solution has a pH of 8.0, the molar ratio of the tris to the NaCl is 1.
By adopting the technical scheme, the buffering range of the Tris buffer solution is 7.1-8.9, and the buffering capacity of the Tris buffer solution is strongest when the pH value is 8.0, so that the pH value of the Tris buffer solution is more than 8.0. A large number of experiments show that the low-glycerol-content protein in the application can form aggregation and is unstable, and the high-concentration glycerol can increase the viscosity of the solution to cause ultrafiltration and chromatographic pressure increase, so that the purification is not facilitated, and therefore, a 5% glycerol solution is adopted. The concentration of sodium chloride is 0.25M, and the concentration of sodium chloride is consistent with the formula of a next chromatography solution, so that the target protein can be completely penetrated in the chromatography process.
Preferably: the ion exchange chromatography in the step (3) comprises the following steps: adopting 20-50mM of trihydroxymethyl aminomethane, 0.15-0.30M NaCl and 0-10% by weight of glycerol aqueous solution to form buffer solution with pH of 7.5-8.5, balancing 5-10 column volumes, loading, and collecting penetrating fluid UV280nm ultraviolet absorption peak, namely the one-step chromatography protein solution.
Preferably: the elution was equilibrated with 50mM Tris, 0.25M NaCl and 5% by weight of glycerol to form a buffer solution of pH 8.0.
Through adopting above-mentioned technical scheme, through a large amount of experiments discovery, adopt low concentration sodium chloride (less than 0.15M) to make a large amount of target proteins adsorb and can't retrieve on the chromatography medium and cause the loss in this application, adopt high concentration sodium chloride (greater than 0.3M) to make a large amount of miscellaneous protein and target proteins pierce through together to reduce the purification effect, consequently adopt 0.25M can the target proteins reach more than 85% recovery rate.
Preferably: the hydroxyapatite chromatography in the step (4) specifically comprises the following steps: and adding 0.5M PB solution into the one-step chromatography protein solution until the final concentration is 25-150 mmol/L PB. 25-150mM PBS and 0-10% by weight of glycerol aqueous solution form buffer solution with pH 6.8-8.0 for balance, sample loading after balancing 5-10 column volumes, collecting penetrating fluid UV280nm ultraviolet absorption peak, namely two-step chromatography protein fluid.
Preferably: the elution was equilibrated with 50mM PBS and 5% by weight of glycerol in water to give a pH8.0 buffer.
Through adopting the technical scheme, a large number of experiments show that a large amount of target protein is adsorbed on a chromatographic medium by adopting low-concentration PB (less than 25 mM) and cannot be recovered, so that loss is caused, and a large amount of impurity protein and the target protein penetrate together by adopting high-concentration PB (more than 125 mM), so that the purification effect is reduced, and therefore, the recovery rate of the target protein can reach more than 85% by adopting 50 mM. Low glycerol content proteins tend to aggregate and become unstable, and high glycerol concentrations tend to increase solution viscosity leading to increased ultrafiltration and chromatography pressures which are detrimental to purification, thus 5% glycerol solutions are used.
Preferably: the hydroxyapatite chromatography in the step (4) specifically comprises the following steps: the two-step chromatographic protein solution is prepared through forming buffering liquid with pH7.5-8.5 with trihydroxymethyl aminomethane in 20-50mM, naCl in 0.15-0.30M and water solution of glycerin in 0-10 wt%, balancing 5-10 column volumes, eluting with PBS in 25-150mM and water solution of glycerin in 0-10 wt%, and collecting UV280nm ultraviolet absorption peak as the eluent.
Preferably: elution was performed with 50mM PBS and 5% by weight glycerol in water to give a pH8.0 buffer.
Through adopting the technical scheme, a large number of experiments show that the target protein adsorbed on the chromatographic medium cannot be lost through elution by adopting low-concentration PB (less than 25 mM), and a large amount of foreign protein and the target protein are penetrated together by adopting high-concentration PB (more than 125 mM), so that the purification effect is reduced, and the recovery rate of the target protein can be up to more than 85% through elution by adopting 50 mM. Low glycerol content proteins tend to aggregate and become unstable, and high glycerol concentrations tend to increase solution viscosity leading to increased ultrafiltration and chromatography pressures which are detrimental to purification, thus 5% glycerol solutions are used.
Preferably: and (3) purifying the two-step chromatography protein solution obtained in the step (4) by using 10-50mM PBS buffer solution with the pH value of 6.8-7.4, 0.1-0.3M NaCl solution and 0.05-3 per mill of Tween-80 in weight-volume ratio, and collecting UV280nm ultraviolet absorption peak to obtain the target protein solution.
By adopting the technical scheme, a large number of experiments show that the protein with the Tween-80 content of less than 0.05 per thousand can form aggregation and is unstable, the detection of the content of the interference protein can be carried out on the high-content Tween 80, and the side reaction can be caused when the high-content Tween 80 is used as an injection additive, so that the better the Tween 80 content is when the process requirements are met.
Preferably: the elution was carried out using 20mM PBS, 0.15M NaCl and 1% w/v Tween-80 solution to prepare a buffer solution of pH7.0.
The invention also aims to provide the application of the purification method of the recombinant CA16 virus-like particles in the preparation of hand-foot-and-mouth vaccines.
The third purpose of the invention is to provide a hand-foot-and-mouth disease vaccine which has good immunogenicity, safety, immune characteristics and biological activity, simple process, no use of ultracentrifuge large-scale equipment and easy large-scale preparation and purification.
The third object of the invention is realized by the following technical scheme: the hand-foot-and-mouth disease vaccine is prepared by purifying recombinant CA16 virus particles according to the purification method in the scheme and then adsorbing the recombinant CA16 virus particles by an aluminum hydroxide adjuvant, wherein the content of CA16 protein is 10-40 mu g/mL, the content of the aluminum hydroxide adjuvant is 0.30-0.60 mg/mL, and the pH value is 6.6-7.4.
By adopting the technical scheme, the prepared vaccine has good immunogenicity, safety, immune characteristics and biological activity, and can not cause inverse toxicity and potential RNA carcinogenic risk. Simple process, no use of large-scale equipment of an ultracentrifuge, and easy large-scale preparation and purification.
Preferably, the cells are disrupted twice by high pressure homogenizing 1250Bar, and the clarified solution is harvested by centrifugation at 7000rpm,40min or by depth filtration; the clear solution is ultrafiltered by a buffer solution formed by 50mm of Tirs and 0.25M NaCl and a 300KD membrane package, and the reflux solution is obtained. The reflux liquid is subjected to ion exchange chromatography, and the collected penetrating fluid is one-step chromatography protein fluid; and then diluting the one-step chromatography protein liquid by using 0.5M PB until the concentration of PB is 50mm, collecting penetration liquid through hydroxyapatite chromatography to obtain a two-step chromatography protein liquid, purifying the two-step chromatography protein liquid by using PBS buffer solution, and collecting UV280nm absorption value to obtain a protein purification liquid.
In conclusion, the beneficial technical effects of the invention are as follows:
1. the invention adopts ultrafiltration and three-step chromatography, has simple operation, strong process controllability and high antigen recovery rate, and is beneficial to large-scale industrial production.
2. The novel vaccine belongs to a recombinant vaccine with extremely high safety, and does not cause inverse toxicity and potential RNA carcinogenic risks. The vaccine of the invention uses aluminum adjuvant, and the immunogenicity of the recombinant CA16 viroid vaccine without the aluminum adjuvant is obviously lower than that of the vaccine containing aluminum adjuvant.
3. The recombinant expression engineering bacteria are subjected to high-density fermentation culture and methanol induced expression of CA16 virus particle protein, the bacteria are centrifugally collected and subjected to high-pressure homogenate crushing, and the supernatant is purified by ultrafiltration, ion exchange chromatography, hydroxyapatite chromatography, molecular sieve chromatography and the like to obtain the recombinant expression CA16 virus particle protein. The CA16 virus particle vaccine provided by the invention has good immunogenicity, safety, immune characteristics and biological activity, is simple in process, does not use large-scale equipment of an ultracentrifuge, and is easier to prepare and purify on a large scale.
4. The vaccine prepared according to the scheme is proved to have strong immunogenicity and good safety through a mouse ED50 experiment and a genotoxicity experiment, can be used for preparing a vaccine for preventing CA16 infection, and has good application prospect.
5. The hand-foot-and-mouth disease caused by the CA16 virus is on the rise, but at present, no vaccine for preventing the hand-foot-and-mouth disease from appearing on the market is available at home and abroad, so that the product can successfully appear on the market and has great social value and economic value.
Drawings
FIG. 1 shows Western blot (Western blot) detection of CA16 strains expressed by engineering bacteria at different time points (0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 hours) after induction, where M is a low molecular weight protein standard (Beijing Quanjin).
FIG. 2 shows the result of HPLC purity measurement.
FIG. 3 is a dynamic light scattering profile of purified CA 16-type virus particles.
FIG. 4 is a transmission electron micrograph (magnification: 98000) of purified CA 16-type virus particles.
Detailed Description
Tris (hydroxymethyl) aminomethane for short
Ethylenediaminetetraacetic acid (EDTA)
Phenylmethylsulfonyl fluoride abbreviated PMSF
Phosphate buffer PB for short
The PBS buffer solution is phosphate buffer solution
A method for purifying recombinant CA16 virus-like particles, comprising the steps of:
(1) Fermenting the recombinant Hansenula polymorpha engineering bacteria containing CA16 coat protein P1 genes and 3CD protease genes;
(2) Crushing engineering bacteria, clarifying and ultrafiltering a target product;
(3) Ion exchange chromatography;
(4) Chromatography on hydroxyapatite;
(5) And (5) performing molecular sieve chromatography.
In the step (1), a high-density formula culture medium is adopted for engineering bacteria fermentation, the bacteria grow rapidly by intermittently supplementing glycerol, an inducer methanol is continuously supplemented for viroid expression, and the engineering bacteria are induced for 36-42 hours in the later fermentation stage. The engineering bacteria fermentation is carried out by adopting a high-density formula culture medium, and the wet weight of the engineering bacteria after the fermentation is finished can reach 250-350 g/L and the antigen expression amount can reach 10 multiplied by 10 through intermittently supplementing glycerin and continuously supplementing inducer methanol 4 ～15×10 4 U/mL。
The step (2) of crushing the engineering bacteria specifically comprises the following steps: the engineering bacteria are adopted in a cell lysis buffer (20 mM NaH) 2 PO 4 ，2mM EDTA-Na 2 0.2-1.0M NaCl,2mM PMSF, 0.01-1.0% Tween-80, pH 6.8-7.4), using a high-pressure homogenizer under pressureThe cell is broken for 2 to 4 times under the condition of 1100 to 1400bar, and the cell breakage rate reaches 80 to 95 percent. Preferably, the engineering bacteria are used for breaking cells for 2 times under the pressure of 1250 bar. The cell disruption rate was 92%.
The clarification of the target product in the step (2) is specifically as follows: pouring the crushed cell sap into a centrifugal cylinder, centrifuging at 6000-8000 rpm for 40-60 min, and collecting clear liquid.
The ultrafiltration in the step (2) is specifically as follows: the collected clear liquid is ultrafiltered by a membrane package of 100-500KD and by using 20-50mM Tris + 0.15-0.30M NaCl + 0-10% glycerin (pH7.5-8.5) to remove small molecular substances, and ultrafiltrate is collected, namely a crude pure product.
The ion exchange chromatography in the step (3) is specifically as follows: taking a Capto Q chromatographic medium as an example, a buffer solution with the pH value of 7.5-8.5 is formed by adopting 20-50mM Tris, 0.15-0.30M NaCl and 0-10% glycerol to balance 5-10 column volumes, then the sample is loaded, and a UV280nm ultraviolet absorption peak of penetrating fluid is collected, namely the one-step chromatographic protein liquid.
The hydroxyapatite chromatography in the step (4) specifically comprises the following steps: adding 0.5MPB solution into the one-step chromatography protein solution until the final concentration is 25-150 mmol/L PB. Using 25-150mM PBS and 0-10% glycerol to form buffer solution with pH6.8-8.0, balancing 5-10 column volumes, loading, collecting penetrating fluid UV280nm ultraviolet absorption peak, namely two-step chromatography protein fluid.
The hydroxyapatite chromatography in the step (4) specifically comprises the following steps: 50mM Tris, 0.25M NaCl and 0-10% glycerol form a buffer solution with the pH value of 6.8-8.0, the buffer solution is balanced for 5-10 column volumes and then is sampled, 25-150mM PBS and 0-10% glycerol solution are adopted for elution, and the ultraviolet absorption peak of eluent UV280nm is collected, namely the two-step chromatography protein solution.
The molecular sieve chromatography in the step (5) is specifically as follows: taking Sephacryl S-300HR as an example, 10-50mM PBS (pH6.8-7.4), 0.1-0.3M NaCl solution and 0.05-3% o Tween-80 are used for purifying the protein solution in two steps, and UV280nm ultraviolet absorption peak is collected to obtain the target protein solution.
The invention also provides a vaccine formulation, each dose of human vaccine comprising:
5-20 μ g of recombinant CA16 virus particles (calculated as protein)
0.15-0.30 mg of aluminum adjuvant (calculated according to Al ions)
The present invention is further illustrated in detail below with reference to tables and examples.
Example 1 fermentation culture of recombinant CA16 Yeast expression Strain in 30L fermentor
The recombinant Hansenula polymorpha strain was inoculated into 100mL of a primary seed medium (0.67% yeast nitrogen source medium, available from SIGMA, 0.5% ammonium sulfate, 2% glucose) and cultured with shaking at 33 ℃ and 200rpm for 20 to 24 hours. Then, the whole amount was inoculated into 1000mL of a secondary seed medium (0.67% yeast nitrogen source medium, 0.5% ammonium sulfate, 2% glycerol), and shaking cultured at 33 ℃ and 200rpm for 20 to 24 hours (OD) 600nm Up to 8-10). Then inoculating the whole amount of the fermented liquid into a 30L fermentation tank, wherein 12L of fermentation medium is filled, the pH value of the fermentation liquid is adjusted by ammonia water and is maintained at 5.0+0.5, the fermentation temperature is 30 ℃, the rotating speed is controlled at 350-750rpm, and the air flow rate is 0.5-1.0m 3 The high-density fermentation needs pure oxygen supplement, the dissolved oxygen is controlled to be 20-60%, when the fermentation medium is 17-21 h, the carbon source is exhausted, the glycerol is supplemented for 2.0L in total, 0.40L is supplemented for 5 times, the glycerol is supplemented when the carbon source is exhausted every time, the dissolved oxygen rises, the thallus growth is about 30-34 h in total, and the wet thallus weight can reach about 0.3-0.4g/mL at most; derepression stage: rotation speed 750rpm, air flow rate 1.0m 3 Controlling dissolved oxygen at 20-60%, adding 1L of mixed solution of glycerol and methanol (200 mL of glycerol and 800mL of methanol) for derepression culture for 34-50h (15-18 h in total); an induction stage: methanol induction is carried out for 50-92h (preferably 36-42 h), and dissolved oxygen is maintained at about 20-40%. And (3) finishing fermentation: when the methanol is completely consumed and the dissolved oxygen is increased to more than 80 percent after 88 to 92 hours, the temperature is reduced to 20 ℃, the fermentation in the tank is ended, and the wet bacteria weight is maintained at 0.25 to 0.35g/mL.
Identification of hansenula polymorpha-expressed CA16 virus-like particles: samples of the above-mentioned cells at various times (0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 hours) after induction were subjected to Western blotting (Western blot) detection, and DAB was developed using an anti-CA 16-VP1 monoclonal antibody (supplied by Beijing Dunbang Biotechnology Co., ltd.) as a primary antibody and HRP-goat anti-mouse-IgG (purchased from Sigma Co.) as a secondary antibody, with the results shown in FIG. 1.
The Western-blot results show that: the expression product can be specifically combined with the monoclonal antibody, and a reaction band which is more obvious for VLP main antigen subunit VP1 protein exists at 33KD, which indicates that the expression product has good immunoreactivity.
And (3) measuring the fermentation expression quantity of the recombinant CA16 virus-like particles: the rabbit polyclonal antibody (obtained by preparing VLP protein immune rabbit by the method of the application and purifying serum) is diluted 1000 times, 100 microliter of the diluted polyclonal antibody is loaded in a 96-hole enzyme label plate hole, and the diluted polyclonal antibody is coated overnight at 4 ℃. Removing the coating solution, and filling the washing solution PBST to wash the ELISA plate. The plate was incubated at 37 ℃ for 2 hours with blocking solution (1% BSA in PBST). And (3) carrying out gradient serial dilution on the supernatant and the reference substance which are centrifugally collected after the crushing, removing the sealing liquid, adding 100 mu L of the sample to be detected and the standard substance into each hole, and incubating for 1 hour at 37 ℃. Washing the microplate 3 times by adding washing solution PBST. 100 μ L of HRP-labeled murine mAb (1 diluted 5000) was added per well and incubated at 37 ℃ for 1 hour. The enzyme labeling solution is removed, and the washing solution PBST is filled to wash the enzyme labeling plate for 3 times. 100. Mu.L of TMB developing solution was added to each well, and the mixture was protected from light at 37 ℃ for 15 minutes. 50 mu L of 2mol/LH is added into each hole 2 SO 4 And (6) terminating. OD determination with microplate reader 450nm And calculating the antigen content by adopting a double parallel line method. The measurement results are shown in table 1.
TABLE 1 detection results of antigen content (ELISA method) of fermentation broke
The results of the ELISA assay showed: the antigen content of CA16 virus-like particles in the fermentation cell disruption solution is 10.8 multiplied by 10 4 U/mL. The CA16 expressed by the hansenula polymorpha cells has higher expression quantity.
Example 2 isolation and purification of CA 16-type Virus particles
Cell collection: the fermentation broth was collected, centrifuged at 6500rpm to collect the precipitate, and the cells were washed twice with cell washing buffer.
Crushing: the collected Hansenula cells were resuspended in cell lysis buffer (20 mM NaH) 2 PO 4 ，2mMEDTA-Na 2 0.2-1.0M NaCl,2mM PMSF,0.5% Tween-80, pH 6.8-7.4), and crushing the cells for 2 times by using a high-pressure homogenizer under the condition of pressure 1200bar, wherein the cell crushing rate is more than 80%.
Clarification: the crushed cell sap is poured into a centrifuge bowl, and is centrifuged at 7000rpm for 40min, and clear liquid is collected. Or filtering the crushed cell sap through a depth filter at a flow rate of 1300mL/min/m 2 And collecting clear liquid night.
And (3) ultrafiltration: and (3) carrying out ultrafiltration on the collected clear solution by using a 300KD membrane package by using 50mM Tris, 0.25M NaCl and 5% glycerol to form a buffer solution with the pH of 8.0 so as to remove small molecular substances, and collecting ultrafiltrate, namely the crude pure product.
Ion exchange chromatography: taking a Capto Q chromatographic medium as an example, a buffer solution with the pH of 8.0 is formed by 50mM Tris, 0.25M NaCl and 5% glycerol and is used for balancing 5 column volumes, then the sample is loaded, and a UV280nm ultraviolet absorption peak of penetrating fluid is collected, namely the one-step chromatography protein fluid. The chromatographic medium is regenerated by using 1mol/L NaOH.
Hydroxyapatite chromatography (transmission mode): adding 0.5MPB solution into the protein solution of the one-step chromatography to the final concentration of 50mmol/L PB. And (3) balancing 5 column volumes by using a buffer solution formed by 50mM PBS and 5% glycerol and having pH of 8.0, then loading the sample, and collecting a UV280nm ultraviolet absorption peak of penetrating fluid, namely the two-step chromatography protein fluid.
Hydroxyapatite chromatography (adsorption elution mode): 50mM Tris, 0.25M NaCl and 5% glycerol form a buffer solution with pH8.0, the buffer solution is balanced for 5-10 column volumes, then the sample is loaded, 50mM PBS and 5% glycerol solution are adopted for elution, and the ultraviolet absorption peak of eluent UV280nm is collected, namely the two-step chromatography protein solution.
Molecular sieve chromatography: sephacryl S-300HR packing is adopted, 20mmPB with pH6.8, 0.15M NaCl solution and 0.1 per thousand Tween-80 are used for purifying two-step chromatography protein liquid, UV280nm ultraviolet absorption peaks are collected to obtain target protein liquid, the purity of the protein liquid is detected to be 100% by HPLC, and the detection result is shown in figure 2.
Detection of the concentration of the protein of interest (Lowry method): accurately measuring standard protein bovine serum albumin solution (200 mu g/mL) 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL, respectively placing the solutions in a test tube, adding distilled water to supplement the solution to 1mL, simultaneously measuring 1mL of 2-fold diluted and purified protein solution in the test tube, respectively adding 5mL of alkaline copper solution and 0.5mL of phenol reagent, and measuring the absorbance value in a cuvette by using a 650nm wavelength. And (3) drawing a standard curve by taking the protein content of the standard protein as an abscissa and the absorbance value as an ordinate, calculating the concentration of the protein solution to be detected, and obtaining a detection result shown in table 2.
TABLE 2 test results of purified protein concentration (Lowry method)
The detection result of Lowry method obtains the final concentration of the purified protein as 163 mug/mL.
Dynamic light scattering analysis of the target protein solution: adding a proper amount of purified CA16 protein liquid into a sample pool, setting the temperature at 25 ℃, the balance time at 90s, setting the automatic cycle times, starting measurement, taking 3 parts of CA16 protein liquid stock solution for parallel test, and analyzing the result. The results show that the recombinant CA16 virus particles are complete, the particle diameters are more than 99 percent in the distribution of 24-30 nm, the PDI is 0.09, and the dynamic light scattering spectrum is shown in FIG. 3.
Analyzing the target protein by a liquid electron microscope: a proper amount of the purified CA16 protein solution was dropped onto a copper mesh, and the mixture was stored in the dark for 5min, after removing excess solution, the mixture was stained with 1% phosphotungstic acid for 2min, and the CA16VLPs were analyzed by Transmission Electron Microscopy (TEM). The results show that the CA16 protein presents virus-like particles, has a regular icosahedral structure of natural viruses, the diameter of the virus particles is about 30nm, and the particles are complete and regular (as shown in figure 4, the magnification is 98000).
And (3) measuring the antigen content of the target protein solution (double-antibody sandwich ELISA): the rabbit polyclonal antibody is diluted 1000 times, 100 mu L of the diluted polyclonal antibody is loaded in the holes of a 96-hole enzyme label plate, and the mixed solution is coated overnight at 4 ℃. And removing the coating solution, and filling washing solution PBST to wash the elisa plate. The plate was incubated at 37 ℃ for 2 hours with blocking solution (1% BSA in PBST). Remove the blocking solution, add 100. Mu.L of sample to be tested into each well, incubate for 1 hour at 37 ℃. Washing the microplate 3 times by adding washing solution PBST. 100 μ L of HRP-labeled murine mAb (1 diluted 5000) was added per well and incubated at 37 ℃ for 1 hour. The enzyme labeling liquid is removed completely,washing solution PBST was added to wash the microplate 3 times. 100. Mu.L of TMB developing solution was added to each well, and the mixture was protected from light at 37 ℃ for 15 minutes. 50 μ L of 2mol/L H was added to each well 2 SO 4 And (6) terminating. OD determination with microplate reader 450nm And calculating the antigen content by adopting a double parallel line method. The results of the double antibody sandwich ELISA assay are shown in table 3.
TABLE 3 double antibody sandwich ELISA test results
Example 3 preparation of recombinant CA16 Virus-like particle vaccine
And (3) main index control: the content of antigen (CA 16 virus-like particles) is 10 mug/mL; controlling the aluminum content at 0.40-0.60mg/mL; the pH value is controlled to be 6.6-7.4.
The preparation method comprises the following steps: the self-made aluminum hydroxide adjuvant is diluted to 0.40-0.60mg/mL by adopting a sterile 0.9% sodium chloride solution. And slowly dropwise adding the purified CA16 stock solution into the adjuvant to ensure that the purified CA16 stock solution is fully adsorbed, and completing the preparation of the vaccine. The detection standards and the detection results for preparing the vaccine are shown in table 4.
TABLE 4 detection results of recombinant CA16 virus-like particle vaccine
| Detecting items | Quality standard | The result of the detection |
| Antigen content | Should not be less than 2500U/mL | 7834U/mL |
| Aluminum content | 0.40-0.60mg/mL | 0.46mg/mL |
| pH value | 6.0-8.0 | 7.0 |
| Adsorption rate | >95％ | 99.5％ |
| Osmolality | 300±65mOSmol/kg | 287mOSmol/kg |
| Bacterial endotoxins | Less than 5EU/mL | Compliance with regulations |
The results in table 4 show that the detection indexes of the recombinant CA16 virus-like particle vaccine all meet the detection standards, and especially, the antigen content is far higher than the minimum standard.
Example 4 immunogenicity of recombinant CA16 Virus-like particle vaccine (ED) 50 ) Test of
Test vaccine: recombinant CA 16-like virion vaccine containing an aluminum hydroxide adjuvant, prepared as in example 3.
Test animals: 50 SPF-grade BALB/c mice, 18-22g, purchased from Experimental animals technology, inc., tokyo Tongliwa, beijing, were selected.
Animal immunization: respectively injecting 10 mice with 0.5mL of vaccine and 0.5mL of aluminum hydroxide adjuvant diluted by multiple times and 0.125, 0.03125 and 0.0078125 mu g/0.5mL of vaccine and aluminum hydroxide adjuvant into abdominal cavity, and removing eyeball and blood at 28 days after immunization. Standing the collected blood at 37 deg.C for 1h, standing at 4 deg.C for 3-4 hr, centrifuging at 4000rpm for 10min, and sucking supernatant.
Detection of neutralizing antibody: serum samples were taken in 2% neonatal bovine serum in MEM culture at 1: diluting at 8 proportion, and inactivating in a 56 ℃ water bath for 30 minutes. A96-well cell culture plate was used, and 50. Mu.L of the dilution was added to each well. Adding corresponding sample 50 μ L into each well, discharging gun, mixing uniformly from line A, sucking 50 μ L to line B, mixing uniformly, diluting sequentially to line D, discarding 50 μ L (serial dilution of CA16 virus challenge virus seed to 100 CCID) 50 0.05 mL), 50. Mu.L of the suspension was dropped into each well, and the cell culture plate was gently mixed and neutralized at 37 ℃ for two hours. The RD cells were digested with the digestion solution to a concentration of 2X 10 5 Adding 0.1mL of cell suspension into each well (including virus back-dripping hole), mixing, and adding 35 deg.C CO 2 Incubating and culturing in an incubator. CPE was observed daily using an inverted microscope and the virus titration results were recorded with the end-point titer being the reciprocal of the highest dilution of serum that inhibited 50% of the cytopathic effect. The final result is judged in 6 to 7 days. The antibody positive conversion rate was shown in Table 5 based on the detection results.
TABLE 5 calculation of antibody Positive conversion rate
Calculated according to the Reed-Muench method: ED (electronic device) 50 ＝0.09(μg)
By mouse ED 50 The experimental results show that: the CA16 virus-like particle vaccine prepared by the invention can ensure that the positive conversion rate of the antibody reaches 50 percent only after 0.09 mu g of immunized mice, so the CA16 virus-like particle has stronger immunogenicity.
Example 5 recombinant CA16 Virus-like particle vaccine Exception test
Vaccine samples: recombinant CA16 virus-like particle vaccine prepared in example 3.
Experimental animals: 10 SPF-rated KM mice (18-22 g) and 4 SPF-rated Hartley guinea pigs (250-350 g) were purchased from Tokyo Wintolite laboratory animal technology, inc.
The experimental method comprises the following steps: before injection, each experimental animal was weighed to 18-22g for mice and 250-350g for guinea pigs. The vaccine was injected into 5 mice and 2 guinea pigs, the mice were intraperitoneally injected with 0.5 mL/mouse, the guinea pigs were intraperitoneally injected with 5.0 mL/mouse, and the observation was carried out for 7 days. A blank control was also set for the same batch of animals. And (4) qualified standard: white control animals and experimental animals in the observation period are kept healthy, abnormal reaction does not occur, and the weight of each animal is increased after the observation period. Animal test conditions are shown in table 6.
TABLE 6 animal Experimental conditions
And (4) conclusion: white control animals and experimental animals in the observation period are kept healthy, abnormal reaction does not occur, and the weight average of each animal body is increased on the 8 th day. The recombinant CA16 virus-like particle vaccine is proved to have no abnormal toxicity, and the safety of the tested animals is better.
Industrial applicability
The invention provides a purification method of CA16 virus-like particles and a method for preparing a vaccine by using the purified CA16 virus-like particles. The recombinant expression engineering bacteria are subjected to high-density fermentation culture and methanol induced expression of CA16 virus granular protein, the bacteria are centrifugally collected and subjected to high-pressure homogenate crushing, and the supernatant is purified by ultrafiltration, ion exchange chromatography, hydroxyapatite chromatography, molecular sieve chromatography and the like to obtain the recombinant expression engineering bacteria. The CA16 virosome vaccine provided by the invention has good immunogenicity, safety, immune characteristics and biological activity, is simple in process, does not use large-scale equipment of an ultracentrifuge, is easy to prepare and purify on a large scale, can be used for preparing a vaccine for preventing CA16 infection, and has good economic value and application prospect.
The above embodiments are given by way of example only and are intended to assist the person skilled in the art in using the invention. The examples are not intended to limit the scope of the invention in any way. Therefore: all equivalent changes made according to the principles of the present invention should be covered by the protection scope of the present invention.
Claims (5)
1. A method for purifying recombinant CA16 virus-like particles, comprising the steps of:
(1) Fermenting the recombinant hansenula polymorpha engineering bacteria containing CA16 coat protein P1 genes and 3CD protease genes;
(2) Crushing engineering bacteria, clarifying and ultrafiltering target product; the ultrafiltration is as follows: ultrafiltering the collected supernatant with 100-500KD membrane package with pH7.5-8.5 buffer solution to remove small molecular substances, and collecting ultrafiltrate to obtain crude pure protein solution, wherein the buffer solution comprises the following components in molar ratio:
2-5 parts of trihydroxymethyl aminomethane
15-30 parts of NaCl
The buffer solution also comprises 0-10% of glycerol aqueous solution by mass fraction;
(3) Ion exchange chromatography;
(4) Chromatography on hydroxyapatite;
(5) Performing molecular sieve chromatography;
the method for crushing the engineering bacteria in the step (2) is to adopt a cell lysis buffer solution for resuspension of the engineering bacteria, and crush the cells for 2-4 times under the condition of the pressure of 1100-1400 bar; the cell lysis buffer was 20mM NaH 2 PO 4 ，2 mM EDTA-Na 2 0.2 to 1.0M NaCl,2mM PMSF,0.01 to 1.0 percent Tween-80, and pH6.8 to 7.4;
the method for harvesting the clarified liquid of the target product in the step (2) comprises the following steps:
pouring the crushed cell sap into a centrifuge tube, centrifuging at 6000 to 8000rpm for 40 to 60min, and collecting clear liquid;
or filtering the crushed cell sap through a depth filter, wherein the filtering flow rate is 925 to 1850ml/min/m 2 Collecting the clear liquid;
the hydroxyapatite chromatography in the step (4) adopts a penetration mode or an adsorption elution mode, wherein:
the hydroxyapatite chromatography penetration mode is as follows: adding 0.5M PB solution into the protein solution of the first-step chromatography until the final concentration is 50mmol/L PB, balancing 5 column volumes by buffer solution formed by 50mM PBS and 5% glycerol and pH8.0, then loading the sample, and collecting UV280nm ultraviolet absorption peak of penetrating fluid, namely the protein solution of the second-step chromatography;
the hydroxyapatite chromatography adsorption elution mode comprises the following steps: 50mM Tris, 0.25M NaCl and 5% glycerol form a buffer solution with pH8.0, the buffer solution is balanced for 5-10 column volumes, then the sample is loaded, 50mM PBS and 5% glycerol solution are adopted for elution, and the ultraviolet absorption peak of eluent UV280nm is collected, namely the two-step chromatography protein solution.
2. The method for purifying recombinant CA16 virus-like particles of claim 1, wherein the ion exchange chromatography of step (3) is: forming a buffer solution with the pH value of 7.5-8.5 by using 20-50mM of trihydroxymethyl aminomethane, 0.15-0.30M NaCl and 0-10% of aqueous glycerol solution by weight percent to balance 5-10 column volumes, then loading, and collecting a penetration solution UV280nm ultraviolet absorption peak to obtain the one-step chromatography protein solution.
3. The method for purifying the recombinant CA16 virus-like particles as claimed in claim 1, wherein the two-step chromatography protein solution obtained in step (4) is purified by using 10 to 50mM PBS buffer solution with pH6.8 to 7.4, 0.1 to 0.3M NaCl solution and 0.05 to 3 per mill of Tween-80 by weight and volume, and UV280nm ultraviolet absorption peak is collected as the target protein solution.
4. Use of the method of purifying recombinant CA16 virus-like particles according to any one of claims 1-3 for the preparation of a hand-foot-and-mouth vaccine.
5. A hand-foot-and-mouth disease vaccine is characterized in that after recombinant CA16 virus-like particles are purified according to the purification method of any one of claims 1 to 3, the vaccine is prepared by adsorption with an aluminum hydroxide adjuvant, the CA16 protein content is 10 to 40 mug/ml, the aluminum hydroxide adjuvant content is 0.30 to 0.60mg/ml, and the pH value is 6.6 to 7.4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019023920A1 (en) * | 2017-08-01 | 2019-02-07 | 金普诺安生物科技（苏州）有限公司 | Foot-and-mouth disease virus-like particle vaccine and preparation method therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019023920A1 (en) * | 2017-08-01 | 2019-02-07 | 金普诺安生物科技（苏州）有限公司 | Foot-and-mouth disease virus-like particle vaccine and preparation method therefor |
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